Project Leader: Zez Williams. This project addresses fundamental gaps in our knowledge of Extracellular RNAs (exRNAs) in circulation and will provide the foundation for understanding both their physiological and pathological functions as well as their potential clinical utility. We currently lack a reference profile of circulating coding and non-coding RNAsand we lack the knowledge of the normal variability to the reference profile within a single individual over time, between similar individuals and between populations differing in gender, body mass index and age. In Aim 1 we will generate a comprehensive and quantitative reference atlas of extracellular RNAs (exRNAs) in circulation from a few healthy individuals and examine the normal variability within an individual and between individuals based on gender, age, and BMl. This atlas will be produce utilizing RNAseq technology developed in our laboratories. This atlas will serve as a foundation for discovery of new biomarkers associated with disease and inform our understanding of potential roles for exRNA in circulation in physiology and disease. We do not understand how different cell- and tissue- types contribute to the total pool of extracellular circulating RNAs. In Aim 2 we will determine the tissue- and cell-specific contribution to total circulating exRNA using the human placenta as a model organ and combining that information with tissue-specific RNAseq data. We will examine which tissues and RNA types contribute to the overall pool of exRNA in circulation. We do not understand the dynamics within the extracellular circulating RNA compartmenthow quickly the component RNAs enter and are cleared from this space. This project seeks to systematically address these areas In Aim 3 we will determine the clearance rate and kinetics of circulating exRNA. This will be accomplished by serially monitoring the plasma and urine of women post-partum and using placenta-specific exRNAs as markers. In summary, we propose to analyze exRNAs in using RNAseq to produce a comprehensive and quantitative reference profile for single individuals over time as well as between populations and to determine the tissue- and cellular origin for these exRNAs as well as their dynamics. Collectively, these results will answer basic questions regarding the full RNA composition of serum, provide a firm foundation for future studies of exRNAs and provide important biomedical insights.